Quanto costa validare un metodo alternativo al ricercatore che lo ha scoperto?

Come diventa obbligatorio un metodo alternativo?

Il ricercatore lo scopre, pubblica i risultati su una rivista scientifica, l’UE sceglie i metodi da validare, inizia l’iter di validazione con costi in buona parte a carico dell’azienda che propone il metodo, il metodo viene validato dopo 10 anni e viene inserito nelle linee guida in modo da diventare obbligatorio.

Quali sono le falle di questo sistema?

1. In primis, è l’Europa stessa a scegliere quali metodi siano da validare. Questa scelta aprioristica automaticamente costringe a scartare diverse potenziali metodologie alternative.
2. La validazione dura un tempo troppo lungo, spesso dunque i metodi validati non sono i più innovativi.
3. I risultati dei metodi alternativi, al posto di essere convalidati con dati conosciuti sull’uomo, vengono comparati con quelli dei test su animali. Considerate le limitatezze dell’animale, l’errore dello stesso può essere scambiato per un risultato giusto, mentre un risultato veramente esatto da parte del metodo alternativo può essere preso per un dato sbagliato (in quanto non concorde con quello dell’animale), conducendo talvolta allo scarto del metodo in fase di validazione.
   Un esempio è quello del Cytosensor Microphysiometer, che doveva rimpiazzare il Draize Test ma diede un risultato inizialmente inconcludente, dato che si confrontò il suo risultato con quello del Draize test stesso, finchè non fu validato grazie a meta-analisi retrospettive, che fecero capire che il problema non era nel metodo alternativo, ma nella presenza di falsi positivi che il Draize test tendeva a dare [1, 2].
4. Spesso non si validano prima i metodi applicabili in più campi, non si dà una priorità ai potenziali replacement nè alle metodiche che utilizzano tessuto umano e non animale.
5. L’Europa fornisce solo limitate risorse economiche per i costi della validazione, tali costi infatti includono la ripetizione di uno stesso esperimento in più laboratori. Una simile spesa dunque è sostenibile da un’azienda, mentre il singolo ricercatore normalmente non riuscirebbe, quindi spesso il paper in cui è descritto il metodo scoperto dal ricercatore va solo ad ingrossare le fila degli articoli di Pubmed, senza poter avere reali possibilità di venire un giorno impiegato come metodica obbligatoria in alternativa di un test animale.
6. Il tutto funziona in ambito regolatorio, mentre per la ricerca di base e applicata non esiste un database di metodi obbligatori costantemente aggiornato, è tutto delegato all’abilità del ricercatore di trovare quello che egli reputa il metodo appropriato di sostituzione (senza che per forza lo sia), se ritiene che vi sia un metodo alternativo.

Per quanto riguarda il punto 5, questa spesa a quanto ammonta? Secondo la dottoressa Meg Lewis dell’azienda biotech “Kirkstall Ltd” (che si occupa di metodi alternativi), come riportato da un paper di ALTEX del 2013, a 5 milioni di Euro per ogni nuovo metodo da validare.

Non è dunque il caso che l’Europa si prenda carico interamente del costo di validazione dei metodi alternativi, e che smetta di limitare l’ingresso all’iter di validazione a metodi scelti aprioristicamente?

Riportiamo l’articolo da cui è preso il dato [3]:

“In the EU, and indeed most other developed countries, the testing of new clinical entities (NCEs) on animals is required by law in order to determine the safety of a new drug before it can be tested on humans. However, the effectiveness of this process is much disputed, given the frequency with which animal testing fails to accurately predict the reactions and interactions of a drug in humans, leading to toxic insult and even death in patients. There are perhaps two explanations for this failure rate – firstly, that the current paradigm of drug discovery is flawed, and secondly that the methodologies employed as part of that paradigm are insufficient to determine the safety and efficacy of new drugs. With each new drug costing an average of $ 6bn to get to market, and with NCE output decreasing annually despite annual R&D spend increases, the pharmaceuticals and CROs are beginning to recognize the limits in cost and effectiveness of current drug discovery approaches.

Alternative methods to traditional testing are required. New EU legislation (Directive 2010/63/EU) states that, where there is an alternative to animal experimentation, that alternative must be used. These methods are on or near to market, and promise to deliver more accurate and sensitive tests for toxicity than animal testing, but early widespread adoption of these new technologies is delayed by the structure of the validation strategy – with an estimated investment of 10 years and € 5m for ECVAM validation of a new assay for example, change is not likely to happen quickly.

If not ethical concern over animal testing, the economic climate is likely to be a key motivating factor in helping drug companies move away from traditional methods and towards new alternatives to current practices, because animal testing is expensive in addition to its limited effectiveness. Alternative approaches promise to reduce the cost of pre-clinical screening, as well as reducing and eventually replacing animal use. Advances in in vitro cell culture technology, combined with quantitative systems pharmacology and new computational approaches to drug discovery, will pave the way for a future of cheaper and more accurate compound testing, better hit rates, and safer and more effective drugs.”

La stessa dottoressa Lewis aveva già riferito in un’intervista [4]:

La validazione rimane un intralcio significativo per l’implementazione di metodi alternativi da parte dell’industria. Con 10 anni e 5 milioni di euro di investimento medio per ottenere un singolo test validato dall’ECVAM, il costo è semplicemente troppo grande per la maggior parte delle piccole imprese, ma senza di essa, è molto difficile convincere le organizzazioni di ricerca a contratto e farmaceutiche ad utilizzare le nuove tecnologie e cambiare i loro approcci (se non impossibile).

Riferimenti bibliografici:

1. [Hartung T, Bruner L, Curren R, Eskes C, Goldberg A, McNamee P, Scott L, Zuang V. First alternative method validated by a retrospective weight-of-evidence approach to replace the Draize eye test for the identification of non-irritant substances for a defined applicability domain. ALTEX. 2010;27(1):43-51.]

2. [Goldberg AM, Hartung T. Protecting more than animals. Sci Am. 2006 Jan;294(1):84-91.].

3. [M. Lewis, J.M. Wilkinson. Moving forward: a new paradigm for drug discovery. ALTEX Proceedings 2, 2/13, LINZ 2013, p. 68.]

4. https://vivoveritas.wordpress.com/2013/09/05/intervista-con-la-drssa-meg-lewis-della-kirkstall-ltd-sul-sistema-quasi-vivo/

Metodi in vitro di barriera epiteliale (pelle, polmoni e intestino) in tossicologia e farmacologia

[Gordon S, Daneshian M, Bouwstra J, Caloni F, Constant S, Davies DE, Dandekar G, Guzman CA, Fabian E, Haltner E, Hartung T, Hasiwa N, Hayden P, Kandarova H, Khare S, Krug HF, Kneuer C, Leist M, Lian G, Marx U, Metzger M, Ott K, Prieto P, Roberts MS, Roggen EL, Tralau T, van den Braak C, Walles H, Lehr CM. Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology. ALTEX. 2015;32(4):327-78.]

Abstract:

“Models of the outer epithelia of the human body – namely the skin, the intestine and the lung – have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.”

Tox21 su Nature: metodi in vitro per sostituire gli animali negli studi di tossicità

[Huang R, Xia M, Sakamuru S, Zhao J, Shahane SA, Attene-Ramos M, Zhao T, Austin CP, Simeonov A. Modelling the Tox21 10 K chemical profiles for in vivo toxicity prediction and mechanism characterization. Nat Commun. 2016 Jan 26;7:10425.]

Abstract:

Target-specific, mechanism-oriented in vitro assays post a promising alternative to traditional animal toxicology studies. Here we report the first comprehensive analysis of the Tox21 effort, a large-scale in vitro toxicity screening of chemicals. We test ~10,000 chemicals in triplicates at 15 concentrations against a panel of nuclear receptor and stress response pathway assays, producing more than 50 million data points. Compound clustering by structure similarity and activity profile similarity across the assays reveals structure–activity relationships that are useful for the generation of mechanistic hypotheses. We apply structural information and activity data to build predictive models for 72 in vivo toxicity end points using a cluster-based approach. Models based on in vitro assay data perform better in predicting human toxicity end points than animal toxicity, while a combination of structural and activity data results in better models than using structure or activity data alone. Our results suggest that in vitro activity profiles can be applied as signatures of compound mechanism of toxicity and used in prioritization for more in-depth toxicological testing.

Mancanza di concordanza nel rilevamento dei non-carcinogeni tra ratti e topi e tra maschi e femmine

[Wang B, Gray G. Concordance of Noncarcinogenic Endpoints in Rodent Chemical Bioassays. Risk Anal. 2015 Jun;35(6):1154-66.]

Abstract:

“Prediction of noncancer toxicologic outcomes in rodent bioassays of 37 chemicals from the National Toxicology Program was evaluated. Using the nonneoplastic lesions noted by NTP pathologists, we evaluate both agreement in toxic lesions across experiments and the predictive value of the presence (or absence) of a lesion in one group for other groups. We compare lesions between mice and rats, male mice and male rats, and female mice and female rats in both short-term and long-term bioassays. We also examine whether lesions found in a specific organ in a short-term test are also found in the long-term test of the same chemical. We find agreement (concordance) across species for specific lesions, as evaluated by the Kappa statistic, ranging from 0.58 (for concordance of nasal lesions between female mice and rats in long-term studies) to -0.14 (lung lesions between mice and rats in long-term studies). Predictive values are limited by the relatively small numbers of observations of each type of lesion. Positive predictive values range from 100% to 0%. Comparing the lesions found in short-term tests to those found in long-term tests resulted in Kappa statistic values from 0.76 (spleen lesions in male rats) to -0.61 (lung lesions in female mice). Positive predictive values of short-term tests for long-term tests range from 70% to 0%. Overall, there is considerable uncertainty in predicting the site of toxic lesions in different species exposed to the same chemical and from short-term to long-term tests of the same chemical.”